It has long been recognized in the medical world that the prevention of preterm delivery or premature rupture of fetal membranes is preferred to the post-treatment thereof. However, a great variety of factors are now known to cause preterm delivery or premature rupture of fetal membranes, making it difficult to prevent these undesirable events. The traditional approach to the prevention of preterm delivery has been identifying a high-risk group of women to which special attention should be paid based on the knowledge of obstetrics and gynecology, demography, and various syndromes (Main et al., Am. J. Obstet. Gynecol., 151:892-898, 1985). However, this approach has the problem of being neither sensitive nor specific. To circumvent this problem, extensive research has been directed to finding biochemical markers for the prediction of impending preterm delivery and premature rupture of fetal membranes, resulting in the nomination of plasma estradiol-17 beta, progesterone, C-reactive protein as promising candidates. However, these candidates were found to be of poor accuracy.
Besides the identification of such biochemical predictable markers, significant attention has been paid to the biochemical role of collagen, based on the fact that the chorionic membrane is composed of fibrous connective tissue and the tensile strength of fibrous connective tissue is determined by its collagen content, as revealed through studies of the premature rupture of fetal membranes. On the basis of their findings that prematurely ruptured fetal membranes have low collagen content compared to normal membranes, some scientists concluded that the premature rupture of fetal membranes is attributable to their low tensile strength compared to that of normal fetal membranes (Obstet. Gynecol., 57:487-89, 1981). According to another study, it has been reported that the serum activity of collagenase was high in prematurely ruptured fetal membranes and preterm labor (Obstet. Gynecol., 75:84-88, 1990). However, the precise mechanism of such biochemical changes has not yet been elucidated (FEBBS Letters, 244(2):315-318, 1989).
Statistically, the frequency of preterm delivery before 37 weeks of gestation is estimated to be about 8 to 10%. In Korea, about 50,000 neonates are prematurely delivered every year. Preterm delivery often causes serious neonatal complications including sepsis, respiratory distress syndrome, pneumonia, bronchopulmonary dysplasia, intraventricular hemorrhage, necrotizing enterocolitis and cerebral palsy. The frequency and severity of such sequelae is greater the earlier the preterm delivery. Therefore, if preterm delivery is prevented, it will be possible to remarkably reduce the occurrence of premature neonates disabled by such diseases.
Recent reports disclose that at least 30 to 40% of preterm deliveries are associated with intrauterine infection (Butler N R., Bonham D G., Prenatal mortality. The first report of the British perinatal mortality survey, Edinburgh, Churchill Livingstone, 115-145, 1963; Romero R., Avila C., Sepulveda W., Preterm birth. Cause, prevention, and management., McGrow-Hill Company, 97-136, 1993; Romero R., Mazor M., Clin. Obstet. Gynecol., 31:553, 1990; Gibbs R S., Romero R., Hiller S L., et al., Am. J. Obstet. Gynecol., 166:1515, 1992).
Intrauterine infection may cause fetal damage by the following process. Intrauterine infection activates the maternal and fetal immune system to secrete inflammatory mediators, such as cytokines from lymphocytes and MMPs (matrix-metalloproteinases) from neutrophils. When the inflammatory mediators reach a certain level, prostaglandin, which promotes uterine contraction, is produced causing active labor leading to preterm delivery. Additionally, increased levels of inflammatory mediators cause the fetus to be affected by fetal inflammatory response syndrome (FIRS). Inflammatory mediators cause sepsis or acute respiratory distress syndrome or damage organs as a result of autoimmune diseases in adults. Likewise, fetal organs can be systemically injured by inflammatory mediators, resulting in brain white matter lesions and bronchopulmonary dysplasia. Therefore, the prenatal diagnosis of intrauterine inflammation is essential for the prevention of preterm delivery and fetal damage.
Generally, the prenatal diagnostic methods of intrauterine fetal infection in current use are cordocentesis, in which fetal blood cytokine levels are measured, and histologic examination of the umbilical cord to identify funisitis. However, cordocentesis is limited in its usage due to its invasiveness, and funisitis can be diagnosed only after delivery (Yoon B H., Romero R., Park J S., Kim C J., Choi J H., Han T R., Am. J. Obstet. Gynecol., 182:675-81, 2000; Yoon B H., Romero R., Kim K S., Park J S., Ki S H., Kim B I., Jun J K., Am. J. Obstet. Gynecol., 181:773-9, 1999; Romero R., Gomez R., Ghezzi F., Yoon B H., Mazor M., Edwin S S., Berry S M., Am. J. Obstet. Gynecol., 179:186-93, 1998).
The white blood cell count in the amniotic fluid is increased in cases of infection or inflammation of the amniotic cavity. Neutrophils in the amniotic fluid are considered to be of fetal origin (Knauper V., Kramer S., Reinke H., Tschesche H., Eur. J. Biochem., 189:295-300, 1990; Blaser J., Triebel S., Massjosthusmann U., Romisch J., Krahl-Mateblowski U., Freudenberg W., Fricke R., Tschesche H., Clinic. Chim. Acta., 244:17-33, 1996; Segura-Valdez L., Pardo A., Gaxiola M., Uhal B D., Becerril C., Selman M., Chest., 117:684-94, 2000; Romanelli R., Mancini S., Laschinger C., Overall C M., Sodex J., MaCulloch C A., Infect. Immun., 67:2319-26, 1999; Maymon E., Romero R., Pacora P., Gomez R., Athayde N., Edwin S., Yoon B H., Am. J. Obstet. Gynecol., 183:94-9, 2000). Therefore, it is postulated that secretory products of neutrophils in amniotic fluid might reflect a fetal inflammatory response. The focus of the present invention is the level of MMP-8 in the amniotic fluid. The determination of MMP-8 in amniotic fluid may be a marker of the fetal inflammatory response syndrome which can be diagnosed currently by histologic examination of the umbilical cord after delivery or by cordocentesis with the determination of fetal blood cytokines.
MMP (matrix metalloproteinase) series, also collectively known as matrixins, are zinc-dependent endopeptidases that function to degrade extracellular matrix proteins. These proteases constitute a large and growing family of proteins which share similar structures and enzymatic properties. MMPs are broadly classified into five groups. Along with MMP-1 and MMP-13, MMP-8 belongs to an interstitial collagenase group. MMP-8 is similar in size to other interstitial collagenases but is glycosylated to a far greater extent. A fully glycosylated proenzyme form of MMP-8 has a molecular weight of 85 kDa. The proenzyme is converted into an active form of 60-70 kDa with the loss of a 15-25 kDa segment. ProMMP-8 is activated in vitro by various proteinases, including trypsin, chymotrypsin and cathepsin G. Organomercurial compounds were also found to activate proMMP-8. The activation mechanism of MMP-8 in vivo has not yet been fully clarified.
The prior technique concerning MMP-8 is found in U.S. Pat. No. 5,736,341, which discloses methods and test kits capable of sensitive and specific diagnosis of periodontal diseases based on monoclonal antibodies against MMP-8. The patent describes that since MMP-8 is directly associated with the destruction of periodontal connective tissues during the progression of periodontitis and diffused into the oral cavity through the gingival pocket containing gingival crevicular fluid, measurement of the MMP-8 level in the oral cavity enables the site-specific diagnosis of periodontitis. For the specific and sensitive biochemical detection of periodontal diseases in progression, these methods measure the conversion of a proform of MMP-8 into an active form because the conversion takes place during the process of periodontal infection. Nothing is mentioned about the use of MMP-8 in connection with preterm delivery and fetal infection and damage.
U.S. Pat. No. 5,641,636 refers to a method of predicting the onset of fetal membrane rupture based on the activity of another matrix collagenase, MMP-9, which belongs to a different group of enzymes from that of MMP-8. MMP-9 is a 92-kDa type IV collagenase/gelatinase or gelatinase B with the largest molecular weight among MMPs. For activation, the proenzyme form of MMP-9, i.e. proMMP-9, is initially cleaved into an intermediate active form of about 83 kDa with concurrent production of a 9 kDa inactive cleavage fragment. The intermediate active form is further processed proteolytically into an active form of MMP-9 with 67 kDa (J. Biol. Chem., 267 (30):21712-21719, 1992). The activation of MMP-9 means the conversion into the 83 kDa intermediate active form or the 67 kDa fully active form with gelatin degradation activity. This patent measures the hydrolytic activity of MMP-9 in degrading denatured collagens, e.g. gelatins, to diagnose the premature rupture of fetal membranes. However, because MMP-9 is already present in the amniotic fluid before the onset of parturition, this method has limited value in predicting the premature rupture of fetal membranes.
Approximately 30 to 40% of patients with preterm labor or premature rupture of fetal membranes undergo preterm delivery. In this condition, various substances, including interleukin-6, interleukin-8, TNF-α, interleukin-1β, GROα, RANTES, white blood cells, MIP-1α, MCP-1, glucose, PGE2, and angiogenin, are known to be present at increased levels in the amniotic fluid. However, these substances are of poor utility for the prediction of preterm delivery because their levels remain unchanged or are not detected in the amniotic fluid of pregnant women without clinical signs of preterm labor and are increased only after the onset of preterm labor or premature fetal membrane rupture.